DNA polymerases are enzymes involved in vivo in DNA repair and replication, but have become an important in vitro diagnostic and analytical tool for the molecular biologist. E. coli DNA polymerase I encoded by the gene “DNA polA” was discovered in 1956, and cloned and characterised in the early 1970s. The enzyme has a variety of uses including DNA labelling by nick translation, second-strand cDNA synthesis in cDNA cloning, and DNA sequencing. The so-called “Klenow” or “Large” fragment of E. coli DNA polymerase I is a large protein fragment originally produced upon cleavage of the native enzyme by the protease enzyme subtilisin. This Large fragment exhibits 5′→3′ polymerase activity and 3′→5′ exonuclease proofreading activity, but loses 5′→3′ exonuclease activity which mediates nick translation during DNA repair in the native enzyme.
Since being discovered in E. coli, DNA polymerase I-like enzymes have been characterised in many prokaryotes, although the non-E. coli counterparts do not always have a 3′→5′ exonuclease proofreading function. Certain DNA polymerase I—like enzymes obtained from various thermophilic eubacteria, for example Thermus flavus, Thermus aquaticus, Thermus brockianus, Thermus ruber, Thermus thermophilus, Thermus filiformis, Thermus lacteus, Thermus rubens, Bacillus stearothermophilus, Bacillus caldotenax and Thermotoga maritima, have been found to be thermostable, retaining polymerase activity at around 45° C. to 100° C.
In general, thermostable DNA polymerases have found wide use in methods for amplifying nucleic acid sequences by thermocycling amplification reactions such as the polymerase chain reaction (PCR) or by isothermal amplification reactions such as strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), and loop-mediated isothermal amplification (LAMP; see Notomi et al., 2000, Nucleic Acids Res. 28: e63). Thermostable DNA polymerases have different properties such as thermostability, strand displacement activity, fidelity (error rate) and binding affinity to template DNA and/or free nucleotides, and are therefore typically suited to different types of amplification reaction.
Isothermal amplification reactions require a DNA polymerase with strong strand displacement activity, and DNA polymerase I enzymes such as Bst DNA polymerase I Large fragment and Bca DNA polymerase I Large fragment are preferred in reactions such as LAMP (see Notomi et al., 2000, supra).
On the other hand, thermocycling amplification reactions such as PCR require a DNA polymerase with reasonable processivity and thermostability at the cycling temperatures used (typically up to 94° C.). Many of the commercially used DNA polymerases for PCR are DNA polymerase II-like enzymes (for example, Vent, Deep Vent, Pwo, Pfu, KOD, 9N7, Tfu DNA polymerases) which lack 5′→3′ exonuclease activity but have proofreading 3′→5′ exonuclease activity. Some DNA polymerase I enzymes (typically those from Thermotoga and Thermus species, for example Taq DNA polymerase) are used in PCR, but Taq DNA polymerase, for example, has insufficient strand displacement activity to function adequately in isothermal amplification reactions.
WO2007/127893 discloses thermostable DNA polymerases from Thermotoga naphthophila and Thermotoga petrophellia. 
Moussard et al. (Int. J. Systemic & Evolutionary Microbiol. (2004) 54: 227-233) discloses the discovery of the genus Thermodesulfatator, with Thermodesulfatator indicus as the type species.
The present invention provides a novel thermostable DNA polymerase I and Large fragment thereof for use in reactions requiring DNA polymerase activity such as nucleic acid amplification reactions. The polymerase, particularly its Large fragment, has surprisingly and advantageously been found to be useful in both thermocycling and isothermal amplification reactions. Included within the scope of the present invention are various mutants (deletion and substitution) that retain thermostability and the ability to replicate DNA.